1. Field of the Invention
The present invention relates to an apparatus and method for detecting endotoxin.
2. Description of the Related Art
All gram-negative bacteria and many fungi have endotoxin as a major constituent of their cell surface. Most gram-positive bacteria have a similar, endotoxin-like molecule as a constituent of the cell surface. In general, bacterial endotoxins partially consist of a highly variable outer region and a conserved inner core. The variable outer region is composed of repeating oligosaccharide (sugar) units comprising the O-ANTIGENIC region. The OUTER CORE and the O-ANTIGEN region, found within the cell walls of various gram negative bacterial species, show species differentiation among endotoxins. Bacterial endotoxins also consist of a relatively constant, conserved inner core region. The conserved inner core consists of the KDO region and the heptose region. The lipid A moiety (also a conserved portion of the endotoxin) is the toxic region of the endotoxin found in the cell surface of gram-negative bacteria. An exemplary endotoxin is shown below: ##STR1##
Endotoxin is an extremely powerful stimulator of the immune system. The devastating effects of bacterial infections and septicemia are in large part due to endotoxin. Mortality rates, due to septicemia are high, 60% or more. The most effective treatment of endotoxin related problems is early detection in real time with high sensitivity. Additionally, endotoxin is a significant contaminant in food products and pharmaceutical products. Accurate determination of endotoxin concentration is required prior to product distribution. To satisfy the requirements of industrial production, endotoxin assays must be accurate, rapid and cost effective.
Levin and Bang observed in the horseshoe crab (Limulus polyphemus) that blood coagulation was a consequence of gram-negative bacterial infections. See Levin, J., Bang, F.B., 19 THROMBOS. DIATH. HAEMORRH., 186-197 (1968), incorporated by reference herein in its entirety and for all purposes. When an extract of the horseshoe crab blood was prepared and tested, a gelation reaction was observed in the presence of endotoxin. Levin and Bang postulated that endotoxin mediated the gelation reaction of horseshoe crab blood. They further postulated that the gelation reaction was initiated enzymatically. The enzyme responsible for initiating the gelation reaction was identified as limulus amebocyte lysate (LAL).
The reaction mechanism for the gelation reaction in horseshoe crab blood involves the activation of a proclotting enzyme by Ca.sup.+ and endotoxin. The activated proclotting enzyme catalyzes the hydrolytic cleavage of coagulogen (a clottable protein of 215 amino acid residues) into polypeptide subunits. Clotting occurs following the cleavage of the 215 amino acid coagulogen protein into a soluble peptide of 45 amino acid residues and an insoluble peptide (coagulin) of approximately 170 amino acid residues. The insoluble 170 amino acid peptide, coagulin, undergoes polymerization to form a stable clot or gel. The presence of a gel or clot indicates the presence of endotoxin. The formation of a gel or clot is used in what is known as the limulus amebocyte lysate method (LAL). The LAL method and its variations are the most commonly used endotoxin detection methods.
The standard LAL tests are of two types, gelation and chromogenic. Both assays are based on the enzyme cleavage reaction of coagulogen, but, in the chromogenic assay, a color is produced during the cleavage step. Thus, the presence of the color instead of a clot or gel indicates the presence of endotoxin.
The limitations of these LAL assays include limitations of specificity, limitations of interfering substances and limitations of reproducibility. The LAL techniques require an enzymatic reaction to detect the presence of endotoxin. Hence, substances that inhibit or stimulate enzymatic cleavage of the 215 amino acid coagulogen protein will lead to false-negative or false-positive results, respectively. In samples, such as serum or blood, there are several factors known to interfere with the LAL method. For example, the LAL enzyme cascade is inhibited by antibiotics, hormones, heavy metals, amino acids, alkaloids, carbohydrates, plasma proteins, enzymes, electrolytes and B-1,3-D-glucan. See Satoshi, M., Masahiro, N., Taizo, W., Tadashi, S. and Tetsuya, T., 198 ANALYTICAL BIOCHEMISTRY 292-297 (1983), incorporated by reference herein in its entirety and for all purposes. For example, false positive gelation can be caused by thrombin, thromboplastin, RNA, RNAase, trypsin, trypsin-like enzymes, lipotechoic acid and peptidoglycan fragments. False negative results (blocking gelation) can be caused by trypsin inhibitors, EDTA, other calcium binding reagents, high salt concentrations and semi-synthetic penicillins. See European Patent No. EP 0 265 127 A1, incorporated herein by reference in its entirety and for all purposes.
Another detection strategy is the sandwich enzyme linked immunosorbent assay (ELISA). This assay (ELISA) involves immobilizing an antibody specific for a conserved region of the endotoxin (e.g. the KDO region). Using ELISA, the endotoxin immobilized (i.e. captured) by a first antibody is detected by using a second antibody attached to another antigenic site of the endotoxin and a chromogenic enzyme. The sensitivity of this technique is about 1 .mu.g/ml. This test is time consuming, requiring over 4 hours.
There are several limitations of the ELISA assay used in the detection of endotoxin. Endotoxin has low affinity for ELISA plates. The lack of endotoxin affinity diminishes sensitivity. When a first antibody is immobilized onto an ELISA plate and endotoxin is bound by (i.e. captured by or immobilized by) the first antibody, there appears to be significant interference with the binding of a secondary antibody to the immobilized endotoxin, used in the detection of endotoxin. Finally, the sensitivity of ELISA assays (in the 1.mu.g/ml range) is far below that of the LAL assays (1 ng/ml for chromogenic LAL assay) and higher than clinically relevant concentrations of endotoxin of about 1 ng/ml.
Other variations of the LAL assays have been developed. These involve combining the LAL assay with an enzyme linked immunosorbent assay (ELISA). A capture antibody (i.e. a polyclonal first anti-endotoxin antibody) for the oligosaccharide region of an endotoxin is immobilized on a microtiter plate. Endotoxin is introduced over the ELISA microtiter plate. The bound endotoxin is detected by using the chromogenic LAL system. Sensitivity is in the range of 2 pg/ml in PBS and 10 pg/ml in diluted plasma. See Mertsola, J., Cope, L.D., Munford R.S., McCracken, G.H. and Hansen, E.J, Detection of Experimnental Haemophilus influenzae Type b Bacteremia and Endotoxemia by Means of an Immunolimulus Assay, 164 THE JOURNAL OF INFECTUOUS DISEASES 353-358 (1991), incorporated by reference herein in its entirety and for all purposes. See Mertsola et al., Specific Detection of Haemophilus influenzae Type b Lipooligosaccharide by Immunoassay, 28 (12) JOURNAL OF CLINICAL MICROBIOLOGY pp. 2700-2706 (December 1990), incorporated by reference herein in its entirety and for all purposes. The combination ELISA/LAL assay requires at least 12-24 hours to complete.
In the combination ELISA/LAL assay systems, the limitations are cumulative. The endotoxin is first bound to an ELISA microtiter plate via a reaction with an antibody or other capture molecule and the endotoxin is then detected using the chromogenic LAL assay. The substances that interfere with the LAL assay also interfere with the combination ELISA/LAL assay. In addition, the LAL reaction portion of the assay requires a minimum of 40 minutes to 2 hours to perform and requires that serum be removed prior to the addition of the enzyme because serum components may inhibit the enzyme activity. the LAL assay does not reliably quantitate the amount of endotoxin present. Detection of endotoxin in serum is five times (5x) less sensitive than detection of endotoxin in buffer.
An antibody-based test reported to have higher sensitivity than ELISA is the latex immunoassay technique. In this assay, latex beads are coated with a monoclonal antibody (Ab1) specific for the O-9 determinant of endotoxin. The beads are then incubated with a solution containing lipopolysaccharide (LPS; synonym for endotoxin). A magnetic bead coated with another monoclonal antibody (Ab2) specific for a different antigenic site of an endotoxin is added to the LPS solution surrounding the latex beads coated with Ab1. In the presence of LPS, the magnetic beads (coated with Ab2) complex with the latex beads (coated with Abl), via the LPS, and the latex beads are sedimented by the use of a magnet. The quantitation of LPS is based on the turbidity of the solution remaining after sedimentation of the magnetic beads (i.e. measuring the latex beads still remaining in solution after sedimentation). The sensitivity varies based on incubation time from 5-30 minutes. Sensitivities of 0.9-25 ng/ml were reported. A serious disadvantage of this assay is that it is inhibited by serum and by high concentrations of endotoxin. See Lim, P., 135 JOURNAL OF IMMUNOLOGICAL METHODS 257-261 (1990), incorporated by reference herein in its entirety and for all purposes.
U.S. Pat. No. 5,057,598 (Pollack et al.) discloses the use of monoclonal antibodies for the immunological detection of endotoxin or endotoxin bearing organisms. See Pollack et al. (U.S. Pat. No. 5,057,598), incorporated by reference herein in its entirety and for all purposes. Pollack et al. states, at column 18, lines 24-28, that detection of endotoxin can be carried out in hours compared with detection of endotoxin based on standard microbiological or cultural methods in days. Clearly, a detection method that works in a time shorter than hours would be advantageous.
The oldest and best known test for endotoxin is the rabbit pyrogen test. This assay has a low sensitivity, is expensive and is plagued with reproducibility problems since different rabbits have different sensitivities to endotoxin challenge. Additionally, animal tests are very time consuming and, therefore, of limited application in a clinical setting.
The immunoassays for endotoxin previously described are all sandwich immunoassays which include the binding of two proteins to the endotoxin molecule. In general, sandwich assays are the preferred approach for the detection of large molecule, whereas competition assays are used for the detection of small molecules with only a single protein binding site. Another sandwich assay for endotoxin, reported by Connelly, uses lipopolysaccharide binding proteins of amebocyte lysates and labelled detection reagents. See U.S. Pat. No. 4,906,567, incorporated herein by reference in its entirety and for all purposes. The general scheme used by Connelly involves holding lipopolysaccharide binding proteins from one or more various organisms (See Col. 4, lines 62-68, U.S. Pat. No. 4,986,567 of Connelly) within the wells of microtiter plates for about 2 hours followed by washing in PBS (phosphate buffered saline), followed by holding BSA (bovine serum albumin) within the same microtiter plate wells for about 1 hour, followed by introduction of an endotoxin containing sample (or sample suspected of containing endotoxin) and holding the sample for about 30 minutes to about 1 hour within the same wells, followed by introduction of a horseradish peroxidase conjugated to an LPS antibody via the heterobifunctional linking agent N-succinimidyl-4-(N-maleimidomehtyl)cyclohexane-1-carboxylate (SMCC) and holding the LPS antibody-peroxidase conjugate within the same wells for for about 30-60 minutes, followed by washing in PBS and introducing a chromogenic substrate, tetramethylbenzidine (TMB) into the same wells and holding for about another 10-15 minutes before taking an optical density measurment at 630 nm. See Connelly at Examples 1, 2, 3, 4 and 5. Id.
In all of the Connelly examples, the pH is maintained at 9.0 or less and the time to reading the optical density (OD) from the time when the sample containing LPS (or suspected of containing LPS) is first intrtoduced into the microtiter plate wells is between about 1 1/6 hours (1 hour, 10 min.--simultaneous or staggered addition of example 5 of Connelly at Col 12, lines 15-35) to about 3 1/6 hours (3 hours, 10 min.--sequential addition of example 4 of Connelly at Cols. 10 and 11).
European Patent (EP 0 265 127) of Harvey and Wilson describes a method and apparatus for the detection of endotoxin using either polymyxin, an octapeptin, or other similar cyclic peptides. An assay is carried out wherein the amount of a polymyxin-endotoxin conjugate (hereinafter, polymyxin B-LPS conjugate) formed is quantitated. The amount of the polymyxin B-LPS conjugate formed is quantitated by attaching a label to either the polymyxin B or to the endotoxin. The labelled polymyxin B-LPS conjugate is then measured. At page 7, lines 27-31 of EP 0 265 127 it is stated that:
In one form of the assay, the analyte which contains LPS and a standard, labelled, LPS preparation compete for a limited amount of immobilized polymyxin B, and the amount of label bound to the polymyxin B is then quantitated. (Emphasis added.) From the above quoted language, it appears at first glance that the analyte (containing LPS or suspected of containing LPS) and the standard, labelled, LPS preparation are simultaneously placed in proximity to the immobilized polymyxin B wherein the analyte LPS and the standard, labelled LPS compete for binding to the polymyxin B. However, upon closer examination of Examples 1, 2, 3, and 4 of EP 0 265 127, it appears that the analyte LPS and the standard, labelled, LPS preparation are not added simultaneously. Instead, the analyte LPS and the standard, labelled, LPS preparation are added consecutively in proximity to the exemplary immobilized polymyxin B. The requirement that the sample and labelled reagent be added sequentially causes the assay to be inherently slower than an assay involving simultaneous addition of analyte and labelled endotoxin. Examples 1-4 describe slow, multistep reactions. PA1 (1) binding capacity of immobilized polymyxin B for LPS is determined by using isotopically labelled LPS (.sup.14 C LPS); PA1 (2) incubating test analyte solution (containing LPS or suspected of containing LPS) with immobilized polymyxin B; PA1 (3) adding a known quantity of isotopically labelled LPS (.sup.14 C LPS) to the mixture of step (2); PA1 (4) measuring the amount of isotopically labelled LPS in solution; PA1 (5) subtracting the amount of isotopically labelled LPS in solution (i.e. unbound isotopically labelled LPS) from the total amount of .sup.14 C LPS introduced in step (3) to determine the amound of .sup.14 C LPS bound to the immobilized polymyxin B; and PA1 (6) subtracting the amount of .sup.14 C LPS bound to the immobilized polymyxin B determined in step (5) from the binding capacity of the immobilized polymyxin B in step (1) to determine the amount of analyte LPS present. The net result of step (6) indicates the amount of analyte LPS present and bound to the immobilized polymyxin B. PA1 (1) incubating analyte LPS samples with a known quantity of polymyxin B alkaline phospatase conjugate in sufficient excess to promote binding between all the analyte LPS and the polymyxin B-alkaline phosphatase conjugate; PA1 (2) incubating the mixture of step (2) with an immobilized, standard LPS preparation to bind the unbound excess of the polymyxin B alkaline phosphatase conjugate from step (1); PA1 (3) rinsing the preparation of step (2); PA1 (4) measuring the amount of the excess polymyxin B-alkaline phosphatase conjugate of step (1) now bound to the immobilized, standard LPS preparation of step (2); and PA1 (5) subtracting the amount of excess polymyxin B alkaline phosphatase determined in step (4) from the total polymyxin B alkaline phosphatase conjugate used in step (1) to determine the amount of analyte LPS present. An assay time of 1 hour plus the time necessary for scintillation counting was required. Sensitivity was 10 .mu.g/ml. The statement was made that increasing the specific activity of the .sup.14 C-LPS would increase sensitivity of the assay. However, increasing specific activity also increases bacground so that the gain from such an improvement in the labelled reagent is rarely greater than a factor of 10. In addition, radiolabels may be hazardous to an inexperienced user and involve undesirable problems of disposal as hazardous waste. PA1 (1) incubating a limited excess amount of immobilized polymyxin B with analyte LPS to bind all analyte LPS; PA1 (2) incubating an excess of standard, LPS-alkaline phosphatase conjugate with the mixture of step (1) and removing excess standard LPs-alkaline phosphatase by rinsing; PA1 (3) measuring the amount of standard LPS-alkaline phosphatase conjugate bound in step (2) to the immobilized polymyxin B; and PA1 (4) subtracting the amount of the immobilized LPS-alkaline phosphatase conjugate bound in step (2) from the total amount of immobilized polymyxin B to determine the amount of analyte LPS bound in step (1). It is difficult to imagine how Example 3 is characterized as a "displacement ELISA" (see p. 10, line 21) since it is stated at p. 8, line 25 that LPS can block the binding of .sup.14 C LPS to polymyxin B/S4B. See EP 0 265 127. PA1 (1) immobilizing analyte LPS and rinsing; PA1 (2) binding polymyxin B-alkaline phosphatase conjugate to immobilized analyte LPS of step (1); and PA1 (3) measuring the amount of labelled polymyxin B attached to the immobilized analyte LPS of step (1) to determine the amount of analyte LPS present in an analyte sample. PA1 Examples 2-4 provide neither data nor experimental details. ELISA assays are generally less sensitive than radioimmunoassays using the same reagents and same general approach. Thus, the approaches described in examples 2-4 would not be expected to produce sensitivity greater than about 10 .mu.g/ml. The use of enzymes as labels also involve problems of interferents from the sample matrix, increasing background response over time during the assay, and increasing instability of the enzyme label during storage. PA1 (1) washing steps are required in Examples 2, 3, and 4; PA1 (2) lengthy incubation steps are required in Examples 1, 2, 3, and 4, so that all assays require more than 1 hour to perform; PA1 (3) a radioactive label is used in example 1; PA1 (4) enzyme labelled LPS or enzyme labelled polymyxin B is used in Examples 2, 3, and 4; and PA1 (5) analyte samples used are extracts from, for example, body fluids (see p. 7, line 33 of EP 0 265 127). (6) no evidence of sensitivity or potential sensitivity greater than .mu.g/ml is provided.
Example 1 of EP 0 265 127 describes a process wherein the following steps are executed:
Example 2 of EP 0 265 127 describes a process wherein the following steps are executed:
Example 3 of EP 0 265 127 describes a process wherein the following steps are executed:
Example 4 of EP 0 265 127 describes a process wherein the following steps are executed:
In all of the assay formats described, European Patent (EP 0 265 127) has several drawbacks:
Thus, there remains a need for an endotoxin assay with high sensitivity to concentrations as low as about 1 ng/ml which can be used in non-homogeneous samples such as serum or saliva, which has no requirement for enzymes or radiolabels, which requires very little manipulation by the operator, which is rapid, which can be used with intact cells, cell fragments or solubilized cells, which can be used on a wide variety of clinical and environmental samples, which requires minimal or no sample preparation, which can be used in a wide variety of environments, structural forms and conditions, which can be used rapidly (between about 15 seconds to about 10 minutes) to test for the presence of endotoxin and which can be adapted to determine the specific type of endotoxin detected.